This invention relates to compositions and methods useful for the prevention, treatment and diagnosis of Lyme disease in humans and other animals. More particularly, this invention relates to OspA and OspB polypeptides which are able to elicit in a treated patient, the formation of an immune response which is effective to treat or protect against Lyme disease. This invention also relates to a screening method for selecting the OspA and OspB polypeptides of this invention which are able to elicit such an immune response. Also within the scope of this invention are antibodies directed against the OspA and OspB polypeptides and diagnostic kits comprising the antibodies or the polypeptides.
Lyme borreliosis is the most common vector-borne infection in the United States [S. W. Barthold, et al., xe2x80x9cAn Animal Model For Lyme Arthritisxe2x80x9d, Ann. N.Y. Acad. Sci., 539, pp. 264-73 (1988)]. It has been reported in every continent except Antarctica. The clinical hallmark of Lyme Disease is an early expanding skin lesion known as erythema migrans, which may be followed weeks to months later by neurologic, cardiac, and joint abnormalities.
The causative agent of Lyme disease is a recently recognized spirochete known as Borrelia burgdorferi, transmitted primarily by ixodes ticks that are part of the Ixodes ricinus complex. B. burgdorferi has also been shown to be carried in other species of ticks and in mosquitoes and deer flies, but it appears that only ticks of the I. ricinus complex are able to transmit the disease to humans.
Lyme disease generally occurs in three stages. Stage one involves localized skin lesions (erythema migrans) from which the spirochete is cultured more readily than at any other time during infection [B. W. Berger et al., xe2x80x9cIsolation And Characterization Of The Lyme Disease Spirochete From The Skin Of Patients With Erythema Chronicum Migransxe2x80x9d, J. Am. Acad. Dermatol., 3, pp. 444-49,(1985)]. Flu-like or meningitis-like symptoms are common at this time. Stage two occurs within days or weeks, and involves spread of the spirochete through the patient""s blood or lymph to many different sites in the body including the brain and joints. Varied symptoms of this disseminated infection occur in the skin, nervous system, and musculoskeletal system, although they are typically intermittent. Stage three, or late infection, is defined as persistent infection, and can be severely disabling. Chronic arthritis, and syndromes of the central and peripheral nervous system appear during this stage, as a result of the ongoing infection and perhaps a resulting auto-immune disease [R. Martin et al., xe2x80x9cBorrelia burgdorferixe2x80x94Specific And Autoreactive T-Cell Lines From Cerebrospinal Fluid In Lyme Radiculomyelitisxe2x80x9d, Ann Neurol., 24, pp. 509-16 (1988)].
B. burgdorferi is much easier to culture from the tick than from humans, therefore at present, Lyme disease is diagnosed primarily by serology. The enzyme-linked immunosorbent assay (ELISA) is one method of detection, using sonicated whole spirochetes as the antigen [J. E. Craft et al., xe2x80x9cThe Antibody Response In Lyme Disease: Evaluation Of Diagnostic Testsxe2x80x9d, J. Infect. Dis., 149, pp. 789-95 (1984)]. However, serologic testing is not yet standardized, and results may vary between laboratories and commercial kits, causing false negative and, more commonly, false positive results. In addition, the disease often goes unrecognized, as the ticks are small and easy to miss, and the characteristic rash only occurs in 60-80% of cases and may be misinterpreted when it does occur.
At present, all stages of Lyme disease are treated with antibiotics. Treatment of early disease is usually effective, however the cardiac, arthritic, and nervous system disorders associated with the later stages often do not respond to therapy [A. C. Steere, xe2x80x9cLyme Diseasexe2x80x9d, New Eng. J. Med., 321, pp. 586-96 (1989).
Two lines of evidence suggest that the host immune response to specific antigens of B. burgdorferi may be partially responsible for the pathogenicity of Lyme disease. First, patients treated with corticosteroids (which suppress the immune system) show improvement of their symptoms [A. C. Steere et al., xe2x80x9cLyme Carditis: Cardiac Abnormalities Of Lyme Diseasexe2x80x9d, Ann. Intern. Med., 93, pp. 8-16 (1980)]. Second, some patients that do not respond to antibiotics appear to manifest an autoimmune disorder initiated by infection with B. burgdorferi. 
Like Trepnema pallidum, which causes syphilis, and leptospirae, which cause an infectious jaundice, Borrelia belong to the eubacterial phylum of spirochetes [A. G. Barbour and S. F. Hayes, xe2x80x9cBiology Of Borrelia Speciesxe2x80x9d, Microbiol. Rev., 50, pp. 381-400 (1986)]. Borrelia burgdorferi have a protoplasmic cylinder that is surrounded by a cell membrane, then by flagella, and then by an outer membrane. Embedded in the outer membrane are two major proteins, a 31 kd outer-surface protein A (OspA) [A. G. Barbour et al., xe2x80x9cLyme Disease Spirochetes And Ixodid Tick Spirochetes Share A Common Surface Antigenic Determinant Defined By A Monoclonal Antibodyxe2x80x9d, Infect. Immun., 41, pp. 795-804 (1983); J. L. Benach et al., xe2x80x9cA Murine IgM Monoclonal Antibody Binds An Antigenic Determinant In Outer Surface Protein A, An Immunodominant Basic Protein Of The Lyme Disease Spirochetexe2x80x9d, J. Immunol., 140, pp. 265-72 (1988)] and a 34 kd outer surface protein B (OspB) [A. G. Barbour et al., xe2x80x9cVariation In A Major Surface Protein Of Lyme Disease Spirochetesxe2x80x9d, Infect. Immun., 45, pp. 94-100 (1984)]. The two proteins have been shown to vary from different isolates or from different passages of the same isolate as determined by their molecular weights and reactivity with monoclonal antibodies. In addition, OspB may not be produced at all in culture [T. G. Schwan et al., xe2x80x9cChanges In Infectivity And Plasmid Profile Of The Lyme Disease Spirochete, Borrelia Burgdorferi, As A Result Of In Vitro Cultivationxe2x80x9d, Infect. Immun., 56, pp. 1831-36 (1988)].
Early in human infection, antibodies are generated primarily against a 41 kd flagella-associated antigen. Later on, high titer antibodies to both OspA and OspB appear [J. E. Craft et al., xe2x80x9cAntigens Of Borrelia Burgdorferi Recognized During Lyme Disease: Appearance Of A New Immunoglobulin M Response And Expansion Of The Immunoglobulin G Response Late In The Illnessxe2x80x9d, J. clin. Invest., 78, pp. 934-39 (1986)]. However, this humoral immune response is generally not sufficient to clear the system of the infective agent in experimentally infected laboratory rats. [K. D. Moody et al., xe2x80x9cExperimental Chronic Lyme Borreliosis In Lewis Ratsxe2x80x9d, Am. J. Troy. Med. Hyg. in press (1990)]. In addition, humans have been shown to be persistently infected for months or years. It has thus been suggested that the spirochete may be able to sequester itself in certain intracellular sites where it remains unavailable to circulating antibody molecules.
Development of a laboratory model for Lyme disease has proved elusive. Several groups have found spirochetemia in rabbits, Peromyscus mice, and Syrian hamsters after inoculation with B. burgdorferi, but no other manifestations of Lyme disease have been found. [W. Burgdorfer, xe2x80x9cThe New Zealand White Rabbit: An Experimental Host For Infecting Ticks With Lyme Disease Spirochetesxe2x80x9d, Yale J. Biol. Med., 57, pp. 609-12 (1984); A. N. Kornblatt et al., xe2x80x9cExperimental Lyme Disease In Rabbits: Spirochetes Found In Erythema Migrans And Bloodxe2x80x9d, Infect. Immun., 46, pp. 220-23 (1984); A. N. Kornblatt et al., xe2x80x9cInfection In Rabbits With The Lyme Disease Spirochetexe2x80x9d, Yale J. Biol. Med., 57, pp. 613-14 (1984); J. L. Benach et al., xe2x80x9cExperimental Transmission Of The Lyme Disease Spirochete To Rabbitsxe2x80x9d, J. Infect. Dis., 150, pp. 786-87 (1984); J. G. Donahue et al., xe2x80x9cReservoir Competence Of White-Footed Mice For Lyme Disease Spirochetesxe2x80x9d, Am. J. Troy. Med. Hyg., 36, pp. 92-96 (1987); E. C. Burgess et al., xe2x80x9cExperimental Inoculation Of Peromyscus spp. With Borrelia Burgdorferi: Evidence Of Contact Transmissionxe2x80x9d, Am. J. Troy. Med. Hyg., 35, pp. 355-59 (1986); P. H. Duray and R. C. Johnson, xe2x80x9cThe Histopathology of Experimentally Infected Hamsters With The Lyme Disease Spirochete, Borrelia Burgdorferixe2x80x9d, Proc. Soc. Exr. Biol. Med., 181, pp. 263-69 (1986); R. C. Johnson et al., xe2x80x9cInfection Of Syrian Hamsters With Lyme Disease Spirochetesxe2x80x9d, J. Clin. Microbiol., 20, pp. 1099-101 (1984).]
Several animal models have been developed however, which suggest that it may be possible to immunize against B. burgdorferi infection. Early studies with hamsters showed that passive immunization, i.e. transfer of serum from rabbits inoculated with B. burgdorferi, conferred protection from subsequent infection with the same strain [R. C. Johnson et al., xe2x80x9cPassive Immunization Of Hamsters Against Experimental Infection With The Lyme Disease Spirochetexe2x80x9d, Inf. Imm., 53, pp. 713-14 (1986)], however this immunity did not extend to strains from other geographic locations [R. C. Johnson et al. xe2x80x9cExperimental Infection Of The Hamster With Borrelia Burgdorferixe2x80x9d, Ann. N.Y. Acad. Sci., 539, pp. 258-63 (1988)]. In addition, active immunization of hamsters with whole inactivated B. burgdorferi also confers immunity, but again it appears to be somewhat strain specific [R. C. Johnson et al., xe2x80x9cActive Immunization Of Hamsters Against Experimental Infection 35 With Borrelia Burgdorferixe2x80x9d, Inf. Imm. 54, pp. 897-98 (1986)]. Hamsters are not an optimal model system however, as they do not appear to develop the clinical symptoms associated with Lyme disease.
An animal model utilizing laboratory rats demonstrated that although they become persistently infected and develop arthritis and carditis, these symptoms are inconsistent if the rats are infected at 3 weeks of age or older [S. W. Barthold et al., supra.]
Another animal model system using the severe combined immunodeficiency (SCID) mouse has also been developed. SCID mice infected with B. burgdorferi contract a chronic infection associated with arthritis and carditis, similar to Lyme disease in humans. [U. E. Schaible et al., xe2x80x9cThe Severe Combined Immunodeficiency Mouse: A Laboratory Model For The Analysis Of Lyme Arthritis And Carditisxe2x80x9d, J. Exy. Med., 170, pp. 1427-32 (1989)]. Using this system, it was shown that B. burgdorferi-specific immune mouse sera as well as a monoclonal antibody to OspA, were able to prevent or slow the development of Lyme disease in SCID mice when passively transferred at the time of infection. [U. E. Schaible et al., xe2x80x9cMonoclonal Antibodies Specific For The Outer Surface Protein A (OspA) Of Borrelia Burgdorferi Prevent Lyme Borreliosis In Severe Combined Immunodeficiency (SCID) Micexe2x80x9d, Proc. Natl. Acad. Sci. USA, 87, pp. 3768-72 (1990)]. However, immunocompromised animals are not well suited for the study of potential vaccines. Others have attempted to infect immunocompetent strains of laboratory mice, but have failed, see S. W. Barthold et al., supra. Thus, additional animal systems and vaccine development is required.
As prevention of tick infestation is imperfect, and Lyme disease may be missed or misdiagnosed when it does appear, there exists an urgent need for the determination of the antigens of B. burgdorferi and related proteins which are able to elicit a protective immune response. In addition, in order to develop agents and methods to prevent and diagnose Lyme disease, an appropriate animal model which mimics the human disease is required with which to study and select such antigens, and to explore the immune response they may confer.
The present invention solves the problems referred to above by providing in one preferred embodiment OspA polypeptides and pharmaceutically effective compositions and methods comprising those polypeptides, which are useful for the treatment or prevention of Lyme disease. The preferred compositions and methods of this embodiment are characterized by OspA polypeptides which elicit in a treated patient, the formation of an immune response which is effective to treat or protect against Lyme disease as caused by infection with B. burgdorferi. 
In another preferred embodiment, this invention provides OspB polypeptides and pharmaceutically effective compositions and methods comprising those polypeptides, which are useful for the treatment or prevention of Lyme disease. The preferred compositions and methods of this embodiment are characterized by OspB polypeptides which elicit in a treated patient, the formation of an immune response which is effective to treat or protect against Lyme disease as caused by infection with B. burgdorferi. 
In yet another embodiment, this invention provides antibodies directed against the OspA or OspB polypeptides of this invention, and pharmaceutically effective compositions and methods comprising those antibodies. The antibodies of this embodiment are those that are immunologically reactive with the OspA or OspB polypeptides of this invention, and are effective to treat or protect against Lyme disease as caused by infection with B. burgdorferi. 
This invention further provides a novel screening process, using a specific nonhuman, mammalian model, for selecting the preferred OspA and OspB polypeptides and antibodies of this invention that are effective to protect against Lyme disease. The screening process of this invention comprises the steps of:
1) immunizing a C3H/He mouse with an OspA or OspB polypeptide or antibody of this invention;
2) inoculating the immunized animal with B. burgdorferi; and
3) selecting those OspA or OspB polypeptides or antibodies which are effective to protect the animal against Lyme disease.
In another embodiment, this invention provides diagnostic means and methods characterized by OspA or OspB polypeptides, or antibodies directed against these polypeptides. These means and methods are useful for the detection of Lyme disease and B. burgdorferi infection. They are also useful in following the course of treatment against such infection.
Finally this invention provides DNA sequences that code for the OspA and OspB polypeptides of this invention, recombinant DNA molecules that are characterized by those DNA sequences, unicellular hosts transformed with those DNA sequences and molecules, and methods of using those sequences, molecules and hosts to produce the OspA and OspB polypeptides of this invention.